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Cereals, including wheat (Triticum aestivum L. em Thell.), are the most important food crops in the world. In addition to use in feed for livestock, the grain from cereals are milled into flour in almost every culture. Wheat flour is found extensively throughout the world but particularly in Europe and North America, rice flour is used extensively in Asia, sorghum flour in Africa, and corn flour (or meal) in the Americas.
The grain of cereal plants varies between species and within species of cereal plants. Grain texture refers to the texture of the kernel (caryopsis), that is, whether endosperm is physically hard or soft. Typically, rice, sorghum, barley and maize are hard textured grains while oat, rye and triticale are soft. Nearly all of the world production and trade in wheat (approximately 550 and 100 mmt annually, respectively) is identified as being either soft or hard. Generally speaking, hard wheat is used for bread whereas soft wheat is used for cookies, cakes and pastries (Morris and Rose, Cereal Grain Quality, Chapman and Hall, New York, N.Y., pp. 3-54 (1996)). The very hard durum wheat (T. turgidum) is generally used in pasta.
In addition to differences in taste and water absorption, grain texture dictates milling techniques. Typically, the harder the grain, the more energy is required for milling, the greater the starch damage during the milling process, and the larger the milled particle size.
A 15 kDa marker protein for grain softness, termed friabilin, is present on the surface of water-washed starch from soft wheats in high amounts, on hard wheat starch in small amounts, and absent on durum wheat starch (Greenwell and Schofield, Cereal Chem. 63:379-380 (1986)). N-terminal sequence analysis of friabilin indicates a mixture of two or more discrete polypeptides (Morris, et al., J. Cereal Sci. 21:167-174 (1994); and Jolly, et al., Theor. Appl. Genet. 86:589-597 (1993)). The two major component polypeptides have been found to be identical to the two lipid binding proteins termed puroindolines (Gautier, et al., Plant Molec. Biol. 25:43-57 (1994)), puroindoline A (puro A) and puroindoline B (puro B), respectively. The transcripts of puro A and puro B, are controlled by chromosome 5D (Giroux and Morris, Theor. Appl. Genet. 95:857-864 (1997)).
Puro A and puro B are unique among plant proteins because of their tryptophan-rich, hydrophobic domains, which have affinity for binding lipids (Blochet, et al., Gluten Proteins 1990, Bushuk and Tkachuk (eds), American Association of Cereal Chemists, St. Paul, Minn., pp. 314-325 (1991); and Wilde, et al., Agric. Res. 20:971 (1993)). The association of friabilin (puro A and puro B) with the surface of the starch granule is apparently mediated by polar lipids. In fact, the occurrence of membrane structural lipids, glyco- and phospho-lipids, with the surface of water washed starch follows that of friabilin (Greenblatt, et al., Cereal Chem. 72:172-176 (1995)): high amounts are present on soft wheat starch, low amounts on hard wheat starch, and none on durum.
There exists a need to modify the texture of grain in cereal plants with more certainty than is available by hybrid crossing. With hybrid crossing, there is the possibility that the parent plants will be reproductively incompatible. There also is the very real possibility that large amounts of water, fertilizer and acreage will be necessary to produce one hybrid plant. By creating transgenic plants with nucleic acid sequences that alter the texture of grain, these problems can be averted. This invention meets this and other needs
This invention provides for the identification of puroindoline A and puroindoline B as the major components of grain softness in wheat (Triticum aestivum). This invention also provides for methods of introducing puroindoline genes and puroindoline homologs into wheat and other cereal plants to modify grain texture.
In a preferred embodiment, a method of producing a transgenic plant with softer textured grain from at least one parent plant with hard textured grain is provided. The method comprises the steps of introducing a nucleic acid sequence which hybridizes under stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3 and operably encodes a puroindoline protein into a cell from the parent plant, and generating a plant from the cell containing the nucleic acid sequence. In a more preferred embodiment, the plant is selected from the group consisting of durum wheat, sorghum, rice, barley and maize. In a most preferred embodiment, the plant is maize. In a preferred embodiment, the introduction of the nucleic acid is mediated by Agrobacterium infection. Also in this embodiment, it is preferred that the puroindoline protein is selected from the group consisting of puroindoline A and puroindoline B.
Another embodiment of this invention provides for a method of producing a transgenic plant with harder textured grain, wherein the plant is derived from at least one parent plant with soft textured grain. The method comprises the steps of introducing a nucleic acid sequence which prevents expression of a puroindoline protein or homolog into a cell from the parent plant and generating the plant from the cell. In a particularly preferred aspect of this embodiment, the plant is selected from the group consisting of wheat, rye, triticale, and oat.
In another preferred aspect of this embodiment the nucleic acid sequence is introduced into a cell and prevents expression of puroindoline A or puroindoline B by operably encoding a ribozyme. In a more preferred embodiment, the nucleic acid sequence operably encodes an antisense nucleic acid which hybridizes under stringent conditions to a nucleic acid sequence complementary to SEQ ID NO:1 or SEQ ID NO:3. Depending on the cereal plant, the nucleic acid can operably encode a transposon. In another aspect of this invention, the nucleic acid sequence is introduced into the plant by Agrobacterium infection.
In yet another embodiment of this invention, a method of producing a transgenic plant with harder textured grain is provided, wherein the plant is derived from at least one parent plant with soft textured grain. The method comprises the steps of introducing a nucleic acid sequence into a cell from the parent plant, wherein the nucleic acid sequence hybridizes under stringent conditions to a nucleic acid as shown in SEQ ID NO:5 and generating the plant from the cell. In a more preferred embodiment, the plant is selected from the group consisting of wheat, rye, triticale and oat. In a preferred aspect of this embodiment, the nucleic acid sequence is introduced into the plant by Agrobacterium infection.
In still another embodiment, a transgenic plant with soft textured grain is provided. The plant is derived from at least one parent plant which has hard textured grain. The plant comprises a nucleic acid sequence which operably encodes a puroindoline protein, wherein the nucleic acid sequence hybridizes under stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3. In a more preferred embodiment, the plant is selected from the group consisting of durum wheat, sorghum, rice, barley and maize. In a particularly preferred embodiment, the plant is maize.
In this embodiment, the puroindoline nucleic acid sequence is introduced into the plant by transformation and the puroindoline protein is selected from the group consisting of puroindoline A and puroindoline B. In a particularly preferred embodiment, transformation is by Agrobacterium infection.
In still another embodiment of this invention, a transgenic plant with harder textured grain is provided. The plant is derived from at least one parent having soft textured grain and comprises a nucleic acid sequence which prevents expression of puroindoline proteins. In a particularly preferred aspect of this embodiment, the progeny plant is selected from the group consisting of wheat, rye, triticale and oat.
In a preferred aspect of this embodiment, the nucleic acid sequence is introduced into a wheat cell and prevents expression of puroindoline A or puroindoline B by operably encoding a ribozyme or a transposon. However, it is preferred that the nucleic acid sequence operably encodes an antisense nucleic acid which hybridizes to a nucleic acid sequence which is complementary to SEQ ID NO:1 or SEQ ID NO:3. In a particularly preferred embodiment, the nucleic acid sequence is introduced into the plant by Agrobacterium infection.
In yet another embodiment, this invention provides for a transgenic plant with hard grain derived from at least one parent having soft grain. The plant comprises a nucleic acid sequence which hybridizes under stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5.
SEQ ID NO:1 is the cDNA sequence of puroindoline A.
SEQ ID NO:2 shows the amino acid sequence encoded by SEQ ID NO:1.
SEQ ID NO:3 is the cDNA sequence of puroindoline B.
SEQ ID NO:4 shows the amino acid sequence encoded by SEQ ID NO:3.
SEQ ID NO:5 is the cDNA sequence of serine substituted puroindoline B.
SEQ ID NO:6 shows the amino acid sequence encoded by SEQ ID NO:5.
SEQ ID NO:7 is a sense strand primer for puroindoline A.
SEQ ID NO:8 is an antisense strand primer for puroindoline A.
SEQ ID NO:9 is a sense strand primer for puroindoline B.
SEQ ID NO:10 is an antisense strand for puroindoline B.
SEQ ID NO:11 is an anti sense strand for serine substituted puroindoline B.
SEQ ID NO:12 is a GSP1 sense strand.
SEQ ID NO:13 is a GSP1 antisense strand.